Figure 1.
Actin isoform expression after liver injury.
In (A–C), stellate cells were isolated after carbon tetrachloride (CCl4) induced liver injury as in Methods and plated on glass coverslips. Twenty-four hours later, smooth muscle α actin (Acta2) (A, Texas red) and nonmuscle β-actin (B, FITC) were detected by immunocytochemistry as in Methods. In (C and D) are shown overlays, revealing co-localization of actins (C: bar = 10 microns; D: bar = 5 microns). Identical results were obtained with cells after either form of liver injury, and images are representative of over 20 others. In (E), stellate cells were isolated from normal livers or 8 days after bile duct ligation or 10 doses of carbon tetrachloride and immediately subjected to immunoblotting as in Methods. Representative immunoblots shown depict duplicate, identical, samples probed for each Acta2 and anti-cytoplasmic β actin (7.5 µg total protein). In (F), specific bands were scanned, quantitated and expressed graphically (n = 4 for each model of injury, *p<0.001 compared to normal). In (G), stellate cells from normal or injured livers were immediately lysed and equal amounts (40 µg) of cellular proteins were subjected to 2-D gel electrophoresis as in Methods. Notably, we also made a theoretical estimation of isoactin PIs by in silico analysis of each actin isoform ([67](Figure S1)). Representative examples (of greater than 20 separate experiments) reveal specific actin isoforms, and after injury (bile duct ligation), new expression of an α isoform (two-D gels are shown in the standard international format with pI ranging from acidic to basic, left to right). In (H), a representative immunoblot of similarly prepared protein samples after 2-D gel electrophoresis is shown (200 µg total protein each). As described in Methods, nitrocellulose membranes were probed sequentially with anti-cytoplasmic β-actin then anti-Acta2 (using the same ECL detection method each time, thus accounting for repeat detection of the β-actin band). Abbreviations: Acta2 - smooth muscle α actin; BDL - bile duct ligation; CCl4 - carbon tetrachloride.
Figure 2.
Enhanced stellate cell motility after liver wounding.
Stellate cells isolated from normal and injured livers were isolated, plated at equal density and allowed to adhere in culture overnight. A linear scratch was applied to the monolayer and cell motility was assessed by phase contrast microscopy (A–B) and by quantitative analysis of cell movement into the scratch-wounded area (C–D). Photomicrographs shown in (A) and (B) depict examples of cells from normal liver (A) and after carbon tetrachloride injury (B) as in Methods; photomicrographs were taken after 24 hours and are representative of 15 different experiments (bar = 60 microns). In (C), cells entering the wounded area of the monolayer over 24 hours were counted (i.e., the number of cells moving the specified distances into the wounded area per high powered field were quantitated as in Methods, n = 6 for each model of injury). In (D), the area in the scratch remaining unoccupied by cells was quantitated (in each experiment, 10 random fields were assessed; the area remaining free of cells was measured by image analysis as in Methods, single data points were created for each experiment and were used to generate quantitative data; n = 6 for each model of injury). For (C) and (D), *p<0.001 compared to normal. Abbreviations: CCl4 - carbon tetrachloride.
Figure 3.
Enhanced migration and contraction of stellate cells after liver injury.
Cells from normal and injured livers were isolated as in Methods and allowed to adhere on top of polyethylene terphthalate membranes containing 8 µm pores. Cells were plated in serum free medium; serum containing medium was placed in the bottom of transwell chambers. After 12 hours, membranes were washed, fixed with 4% paraformaldehyde and stained for 30 minutes with 0.4% hematoxylin. In (A) and (B) are shown representative examples of cells from normal liver and in (C) and (D) are shown cells from injured liver (carbon tetrachloride). Panel (A) shows an exposure focused on the top of the membrane, (B) depicts the same field, but focused on the bottom of the membrane. In (A), many cells remain compact and therefore are darkly stained, the small arrows point to cells that have begun to spread on the top of the membrane. In (B), no cells have passed through the membrane and therefore none are in focus. In (C) and (D) virtually all cells have spread markedly, the small arrows in (C) point to cells that have spread on the top of the membrane. In (D), the larger arrows point to cells that have migrated through the membrane (bar = 50 microns). In (E), the number of cells migrating to the bottom of the membrane were quantitated and expressed as a proportion of all cells plated (n = 4 for each model of injury, *p<0.001 vs. control (normal cells)). In (F), stellate cells from normal and injured livers were isolated and allowed to adhere on top of collagen lattices. After adherence for 18 hours, serum free conditions were introduced and medium containing endothelin-1 (2 nM) was added. Lattices were dislodged and contraction after 4 hours is shown (n = 4 for each injury model, *p<0.001 vs. control (normal cells)). Abbreviations: BDL - bile duct ligation; CCl4 - carbon tetrachloride; Nl - normal; Ctr – control.
Figure 4.
Acta2 expression in normal and injured stellate cells during cell migration.
Stellate cells from normal and injured liver (carbon tetrachloride) were isolated, plated at equivalent density and allowed to adhere in culture overnight as in Figure 1. After 12 hours, a linear scratch was applied to the monolayer. Twenty-four hours later, cells were fixed and dual labeled with anti-cytoplasmic β-actin and anti-Acta2 antibodies as in Methods. In (A, cytoplasmic β-actin) and (B, Acta2), representative examples of cells from normal livers after scratch wounding are shown. In (D, cytoplasmic β-actin) and (E, Acta2), cells from carbon tetrachloride treated animals are shown. In C and F, co-localization of β-actin and Acta2 is depicted in overlays. Representative areas from typical experiments (carbon tetrachloride) are shown (n = 15) (bar = 100 microns).
Figure 5.
Acta2 antisense oligodeoxynucleotides inhibit Acta2 expression, stellate cell contractility, and stellate cell motility.
Stellate cells were isolated from normal rat livers; after 24 hours, oligonucleotides were transfected as in Methods (the transfection mix containing oligonucleotides was replaced every 48 hours). Five days later, cells were harvested and lysates were subjected to immunoblotting to detect Acta2. In (A), different oligonucleotides (10 µM) were tested; specific Acta2 bands were scanned, quantitated and expressed graphically (n = 3, * p<0.01). In (B), the effect of different concentrations of sense and antisense oligonucleotides (the Acta2 3′UT #1 sequence) was examined. The upper portion of the figure depicts a representative immunoblot, and the graph below depicts scanned and quantitated data (n = 3, * p<0.01). Immunoblots with anti-cytoplasmic β-actin revealed no change in Acta2 expression (not shown). In (C), cells as above were placed on collagen lattices; oligonucleotides were added 24 hours later (all at 10 µM) and replaced at day 3 and 5 in culture. Serum free conditions were introduced and medium containing serum (10% horse/10% calf) was added to induce contraction. Lattices were dislodged from their plastic substrata and gel contraction was measured (contraction after 4 hours is shown, n = 4, *p<0.01 compared to lattices exposed to sense oligonucleotides). Cells exposed to only serum free or serum containing medium served as negative and positive controls, respectively. In (D–H), stellate cells from normal livers were isolated and allowed to undergo culture induced activation. Twenty-four hours after isolation, cells were transfected with oligodeoxynucleotides as in Methods. Seventy-two hours later, a linear scratch was applied to the cell monolayer. In (D), cells exposed to 3′UT #1 sense oligonucleotides (10 µM) are shown; in (E) cells exposed to 3′UT #1 antisense oligonucleotides (10 µM) are shown (representative images 24 hours after scratch wounding are shown) (bar = 50 microns). In (F), the number of cells per high-powered field entering the wounded area of the monolayer were counted and quantitated as in Methods (n = 6, *p<0.01 vs. cells exposed to sense oligonucleotides). In (G), the area in the wound remaining unoccupied by cells was quantitated by image analysis as in Methods (n = 6, *p<0.01 vs. cells exposed to sense oligonucleotides). In (H), the effect of Acta2 antisense oligodeoxynucleotides on stellate cell motility was assessed by measuring migration of stellate cells through polyethylene terphthalate membranes containing 8 µm pores as in Figure 2 (n = 3, *p<0.01 vs. to sense). Abbreviations: Init - initiation; UT – untranslated.
Figure 6.
Reduced cellular motility and contractility in Acta2 deficient cells.
Acta2 wild type (+/+) and null (−/−) fibroblasts were isolated from mouse embryos as in Methods. At the second to sixth passage, cells were plated in monolayers at uniform density and subjected to scratch wounding as in Methods. In (A) (+/+) and (B) (−/−), representative examples of cells migrating into scratched areas at different times are shown. In (C), cells migrating the specified distances and 12 and 24 hours after scratch wounding were counted (n = 6, *p<0.01 for +/+ vs. −/− cells). In (D), stellate cells from Acta2 deficient (−/−) and wild type (+/+) were placed on top of collagen lattices and contraction was measured as in Methods (n = 4, **p<0.005 for +/+ vs. −/− cells).
Figure 7.
In (A), rat stellate cells were isolated and grown in standard medium for 2 days as described in methods and then exposed to smooth muscle (SM) α actin (Acta2) siRNA (siActa2) or control siRNA (siLuc) for 48 hours as in Methods. Cells were incubated in 0.5% serum medium for a further 24 hours and then harvested. Equal quantities of protein lysate (25 µg) were subjected to immunoblotting to detect the identified proteins and representative images are shown; quantitative data are presented graphically (B and C, n = 3; *p<0.05 for siLuc vs. siActa2). In (D), stellate cells as above were seeded at a density of 1×104 per well in 96 well plates and transduced siRNA siActa2 or control siRNA siLuc for 48 hours and then incubated in 0.5% or 10% serum medium for a further 24 hours. Cell proliferation was measured as described in Methods, with proliferation being proportional to absorbance. Abbreviations: SM - smooth muscle; siActa2 - smooth muscle α actin or Acta2 siRNA; siLuc - luciferase siRNA.